The hsp90s are a family of cellular chaperones that are responsible for the maturation of a diverse set of client proteins including transcription factors, proto-oncogenic kinases, immune response mediators, and many others. The transformation of the proto-oncogenic kinase v-Src was shown to be highly dependent on hsp90s. Small molecule inhibitors, which are now under therapeutic development, disrupted this maturation. The anti-tumor activity of these compounds was attributed to the inhibition of oncogenic client release by hsp90, which targeted these complexes for ubiquitin-dependent degradation. Structural studies have shown that these inhibitors bind competitively to the ATP-binding pocket in the N-terminal domain of hsp90s. The activity of hsp90s is linked to cycles of ATP binding and hydrolysis. There is a gap, however, in our understanding of the process that relates ATP hydrolysis and client maturation. Despite their high sequence homology, mammalian and lower eukaryotic members of the hsp90 family exhibit significantly different rates of ATP hydrolysis. These correlate with conformational differences seen in recent crystal structures of intact mammalian GRP94, the ER hsp90 paralog, and yeast Hsp90. Using biochemical assays of ATPase activity in conjunction with point mutants, domain swaps, and in vivo functional studies, I propose to dissect the mechanistic differences between members of the hsp90 family in order to understand what elements of each hsp90 paralog contribute to the regulation of its ATPase activity. In addition, I propose to determine the crystal structure of intact human Hsp90, another slow ATPase, to test the hypothesis that slow rates of hydrolysis derive from a conformation similar to that observed for GRP94, and that all higher eukaryotic hsp90s adopt this or similar conformations. Finally, I propose to assemble complexes between GRP94 and potential client proteins in order to probe the basis for client protein interactions in this system. These studies will help further our understanding of the ATPase activity in hsp90 by relating it to distinct structural elements, and will also provide insight into client-hsp90 interactions. Hsp90 chaperones are the targets of a new class of anti-cancer drugs. Our ability to safely use these drugs, and develop better ones, depends on a detailed understanding of how these hsp90 chaperones work. I propose to dissect the mechanism of hsp90 chaperones in order to understand how each of the four different forms of hsp90 function in the cell. This may ultimately lead to the design of new classes of hsp90 drugs that are useful for the treatment of other diseases as well, such as sepsis and stroke.